Integrity of proteins in reconstituted chromatin

Vol. 67, No. 4, 1975

BIOCHEMICAL

INTEGRITY Chi-Born

Chae,

Received

A. Gadski,

of Biochemistry, Chapel Hill,

October

RESEARCH COMMUNICATIONS

OF PROTEINS IN RECONSTITUTED CHROMATIN

Robert

Department

AND BIOPHYSICAL

Donald

B. Carter

University North Carolina

and Peggy H. Efird

of North 27514

Carolina,

28,1975

SUMMARY. Rat liver chromatin reconstituted from fractionated histones, chromosomal non-histone proteins, and DNA is extensively degraded by chromatin-bound protease. Chromatin proteins

reconstituted

quite

and specific

with

the

studies

important

to its

This specific

on the

into

DNA and

of tissue-specific that

suggests

manner

on the mechanism

information

dissociation

transcription

RNA (5,6).

DNA in a highly

systematic

duce very

mediates

messenger

reassociate fore,

often

subsequent

chromosomal

during

components

reconstitution.

of chromatin

regulation

PSJA (l-4)

There-

reconstitution

of genes

will

pro-

at the chromosomal

level. Our preliminary were

very

disappointing,

proteins

during

We have

found

protease

that

non-histone identical

this bound

and Toczko

However,

Since

isolated

of the reported by the method

and urea

thus

rendering

degradation

is

(lo),

and his

similar

(13)

reported

tissues

of chromosomal of salt

due to a small

a type

rat

group

of the liver

of chromatin

methods

degradation

rat

and urea

molecular

(7-9).

weight

to the enzyme reported

12).

proteins

method

from several

in the presence

to chromatin

of native

prepared

after

protein

Stein

chromosomal

their

chromatin

reconstitution

(11,

recently

to those

with

due to the proteolytic

chromatin

tightly

by Kurecki

most

studies

a faithful

et al.

rat

the histones

liver

chromatin

and are

chromatin. preparation

we have

of Stein

reconstituted

that

decided is

replica

reconstitution.

1459

is

somewhat

to determine

devoid of the

if

different the

of the protease initial

from

chromatin active

chromosomal

in salt proteins

Vol. 67, No. 4, 1975

MATERIALS

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS

Preparation of chromatin - Chromatin was prepared from fresh liver of male Sprague-Dawley rats (average weight 200 g) according to the method of Stein et al (13) as follows: Fresh livers were homogenized in 3 vol. (V/W) of icecold 0.25 M sucrose - 0.05 M KC1 - 5 mM MgC12 - 50 mM Tris (pH 7.4) by a PotterElvehjem homogenizer with a motor-driven Teflon pestle and filtered through six layers of cheesecloth and one layer of miracloth. The homogenate was centrifuged at 2000 g for 5 min. The pellet was washed three times with Earle's balanced salt solution (without phenol red) by homogenization in a loose-fitting Dounce homogenizer and subsequent centrifugation at 2000 g. The crude nuclear pellet was then washed three times in 80 mM NaCl - 20 mM EDTA 1% Triton X-100 (pH 7.0) by homogenization in a loose-fitting Dounce homogenizer and subsequent centrifugation at 1000 g. The nuclei were then washed three times with 0.15 M NaCl - 0.01 M Tris (pH 8) and suspended in cold deionized water. Chromatin was swelled for 10 min and sheared in a Vir Tis homogenizer at 30 volts for 1 min and the concentration of chromatin was adjusted to 20 A2b0 units/ml. All procedures were carried out at 0-4OC. Dissociation of Stein et of 10 A260 dissociated supernatant

of chromatin - Chromatin was dissociated according al (13) in 3 M NaCl - 5 M urea - 10 mM Tris, pH 8 at units/ml (Total volume 50 ml) by stirring at 4'C for chromatin was centrifuged at 150,000 g for 48 hrs at and DNA-pellet were saved.

to the method a concentration 4 hrs. The 2'C, and the

Fractionation of histones and non-histone chromosomal proteins - The supernatant containing histones and non-histone proteins was fractionated into histones and non-histone proteins according to the method of Gilmour and Paul (14) as described by Stein et al (13): The supernatant was saturated with solid ammonium sulfate (Schwartz/Mann, The precipitate was collected by Enzyme Grade) and stirred for 2 hrs. The protein pellet was suspended in centrifugation at 50,000 g for 30 min. 10 ml of 5 M urea - 10 mM Tris, pH 8 and dialyzed against 500 ml of 5 M urea The 5 M urea solution was changed 3 times over a 10 mM Tris, pH 8 overnight. 12 hr period. The dialyzed protein was mixed with a 40 ml packed volume of QAR-Sephadex which was titrated to pH 8 and equilibrated with 5 M urea - 10 mM was stirred at OoC for 10 min and filtered through a Tris, pH 8. The mixture The filtrate (histones) was saved. The QAE-Sephadex sintered-glass funnel. protein was washed with 200 ml of 5 M urea - 10 mM Tris, pH 8. Non-histone fraction was then eluted from the QAE-Sephadex with three lo-ml portions of and non-histone protein 3 M NaCl-5 M urea - 10 mM Tris, pH 8. The histone fraction were precipitated with ammonium sulfate, and the precipitate was collected by centrifugation at 50,000 g for 30 min. The whole procedure from the high-speed supematant of dissociated The chromatin to QAE-Sephadex fractionation of proteins took two days. final yield of histones and non-histone proteins were 7 and 3 mg, respectively, from chromatin containing 25 mg DNA. Reconstitution of chromatin - DNA (pellet obtained from the high-speed centrifugation of dissociated chromatin), histone, and non-histone proteins were dissolved separately in small volumes of 3 M NaCl - 5 M urea - 10 mM Tris, pH 8 and dialyzed against the same solution for 3 hrs. DNA, histone, and non-histone protein fraction were combined at the ratio of 1: 2:2 (W/W) as described by Stein et al (13) and dialyzed consecutively 4 hrs each against 5 M urea - 10 I& Tris, pH 8 containing 2 M NaCl, 1.5 M NaCl, 1 M NaCl (overnight), 0.6 M NaCl, 0.4 M NaCl, 0.2 M NaCl, 0.1 M NaCl (overnight) and finally 0 M NaCl. The final mixture in 5 M urea - 10 mM Tris, pH 8 was

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Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

centrifuged at 20,000 g for 30 min, and the supematant and pellet In Stein's procedure the pellet fraction for gel electrophoresis. dialyzed against 10 mM Tris, pH 8 (13).

were saved was further

Sodium dodecyl sulfate (SDS) gel electrophoresis - Native chromatin and various fractions obtained above were dialyzed against 1% SDS - 0.01 M sodium phosphate (pH 7) - 0.1% 2-mercapto ethanol at room temperature, and the proteins were electrophoresed through SDS polyacrylamide (7.5%) gels as described before (7). RESULTS AND DISCUSSION As shown weight

in Figure

non-histone

chromatin. fraction

proteins

The degradation (F2al)

1 there

is

also

was considerable

degradation

of high-molecular

at the end of 48 hr centrifugation product

apparent.

migrating The histone

faster fraction

than

of the the smallest

obtained

from

dissociated histone the QAE-

Figure 1. SDS electrophoretic patterns of chromosomal proteins during dissociation and reconstitution of chromatin. (1) Native chromatin; (2) DNA pellet and (3) supernatant after centrifugation of dissociated chromatin; (4) total proteins after ammonium sulfate precipitation and dialysis against 5 M urea - 10 mM Tris, pH 8; (5) histone and (6) nonhistone protein fraction obtained from QAE-Sephadex chromatography; (7) reconstituted chromatin and (8) supematant after centrifugation of the chromatin dialyzed against 5 M urea - 10 mM Tris, pH 8; and (9) native chromatin.

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Vol. 67, No. 4, 1975

Sephadex

BIOCHEMICAL

chromatography

obviously

lacks

extensive

degradation.

the native

are

We also

control

into

total

that

as far

against Chromatin

of chromatin Also,

remains

in

chromatin

does not

overall

electrophoretic

it

recovered

soluble

in 5 M urea,

at 20,000 is

complex

- 10 mM Tris,

is

g will

obvious

the supernatant

Degradation

from after

not

Figure

recover 1 that

of Incubation

Acid-Soluble

resemble patterns

pH 8 contains and, all

only

therefore, the

the

chromatin

a considerable

amount

in

Radioactivity (cpm>

released

500 480

16 hr

2180

4OC, 16 hr

1630

Chromatin

(A260 =15)inlmlof3MNaCl-5Murea-lOmMTris,

pH 8 was mixed

with

3000 cpm) labelled 0 hr or 16 hr

supematant

at all

by a low centrifugation

Protease

0 hr

ice-cold

and shows

1

0 hr

Room temperature,

for

fraction

centrifugation.

of Histones by Chromatin-Bound 3 M NaCl - 5 M urea

Room temperature, 4'C,

proteins

5 M urea

Table

Condition

non-histone

as the

the DNA-protein

DNA.

a pellet.

protein

weight

protein

concerned.

found

centrifugation

The non-histone

The reconstituted

at the end of the dialysis 30% of the

Fl histone.

the high-molecular

chromatin

of proteins

lacks

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

170 pg of with

r3~]

[3H1 Fl + F2b histones acetic

anhydride.

at 4oC or at room temperature,

50% trichloroacetic was determined

acid, after

and radioactivity centrifugation

1462

(Total

radioactivity,

The chromatin, was mixed

with

incubated 1 ml of

in the acid-soluble of the precipitate.

of

BIOCHEMICAL

Vol. 67, No. 4, 1975

That

the protease

active

chromatin

was tested

histones.

As shown

in Table

degrades

labeled

et al

(13)

temperature.

have

found

that

methane

sulfonyl

results

suggest

Labelling

the

dalton

tissues

1 the

protein

is

tissues

(70,000, DFP-binding

We have partially

fractionated

radioactive

(DFP) a serine with

60,000

of Stein

methods

can be inhibited

is

liver

at 40 and room

by other

protease

rat

by the chromatin

fluorophosphate

from various

the major

produced

activity

chromatin-bound

weights

from

by the method

prepared

protease

prepared

peptides

prepared

peptide

and diisopropyl

the

in Stein's

in 3 M NaCl - 5 M urea

same as by chromatin

molecular

(10).

chromatin

RESEARCH COMMUNICATIONS

was present

of acid-soluble

of acid-soluble

fluoride that

and urea

histones

chromatin-bound

of different

25,000

various

the

of chromatin

proteins

in salt

by the release

The amount

seems to be about

AND BIOPHYSICAL

(9).

We

by phenyl(9).

These

protease.

3H-DFP shows three and 25,000).

protein this

Of these,

in chromatin small

from

molecular

--

Rf Figure 2. Labelling patterns of chromosomal proteins by [3H]-diisopropyl fluorophosphate. Chromatin (A260 = 15) in 1 ml of 10 r&I Tris, pH 8 was mixed with 5OuCi of [1,3-3H] DFP (2 Ci/mmole, Ammersham/Searle) and incubated overnight at room temperature. At the end of incubation the chromatin was made 1% SDS-O.01 M sodium phosphate-0.1% 2-mercaptoethanol and dialyzed exhaustively against the same solution. The chromatin was electrophoresed through a SDS polyacrylamide (7.5%) gel and 1.5 nnn slices were counted as described by Carter et al (10).

1463

Vol. 67, No. 4, 1975

weight in

protease

BIOCHEMICAL

from

also

contains

Therefore,

it

The chromatin

affected

X-100 balanced

salt

solution,

and urea

very

distressing

field

chromosomal

chromatin

to us.

not

reconstitution proteins.

in reconstituted by various

protease

RNase-free

absence

in chromatin

be routinely

2. for

or heated), red

and deionized

the work for

compared

Triton

in Earle's grade)

were

protease.

of Stein

et al

investigators

successful

the

is not

preparation:

of phenol

that

in Figure

and reconstitution

of proteolytic

we recommend

of Stein

(13).

grade,

are

active

responsible

in chromatin

may be useful

laboratories

in the

chromatin

it

also

of chromatin-bound

to duplicate

all

Therefore,

grade

activity

However,

that

weight

(reagent final

is

et al

used

highly

as shown

dissociation

and presence

we are not able

to be aware

chromatin

proteins

that

on the

protease

of Stein

reagents

is

by the method

protease

during

molecular

absence

sources,

protease

prepared

dalton

by the method

grade,

this

weight

proteins

(reagent

to have no effect The fact

the 25,000

of various

sucrose

that

molecular

of the small

from various

found

that

by the purity

For example,

small

prepared

The activity

out

(10).

of chromosomal

of the chromatin

this

and found

this

appears

the degradation

is

chromatin

3 M NaCl - 5 M urea

et al

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

in carrying

degradation integrity with

(13)

of

of chromosomal those

of native

methods.

ACKNOWLEDGEMENTS This (CA-17390) N.I.H.

work was supported by grants from National and National Institute of General Medical

Cancer Institute Sciences (GM-21846),

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Bekhor, I., Kung, G. M. and Bonner, J. (1969) J. Mol. Biol. Huang, R. C. C., and Huang, P. C. (1969) J. Mol. Biol. 2, Gilmour, R. S. and Paul, J. (1970) FEBS Lett. 2, 242-244. Spelsberg, T., Hnilica, L. and Ansevin, T. (1971) Biochim. 228, 550-562. Barrett, T., Maryanka, D., Hamlyn, P. H., and Gould, H. J. Nat. Acad. Sci., U. S. 71, 5057-5061. Paul, J., Gilmour, R. S., Affara, N., Bimie, G., Harrison, Humphries, S., Windass, J. and Young, B. (1973) Cold Spring Quant. Biol. 3, 885-890. Chae, C-B., and Carter, D. B. (1974) Biochem. Biophys. Res. 740-746. Chae, C-B (1975) Biochemistry 16, 900-906.

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2, 351-364. 365-378. Biophys. (1974)

Acta Proc.

P., Hell, A., Harbor Symp. Commun. il_,

Vol. 67, No. 4, 1975

9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Carter, D. B. and Chae, C-B. Biochemistry, in press. Carter, D. B., Efird, P. H., and Chae, C-B. Submitted. Kurecki, T. and Toczko, K. (1972) Bulletin De L'Academic Polonaise Des Sciences. 2, 543-546. Kurecki, T. and Toczko (1974) Acta Biochimica Polonica 1_1_, 225-233. Stein, G. S., Mans, R. J., Gabbay, E. J., Stein, J. L., Davis, J. and Adawadkar, P. D. (1975) Biochemistry 16, 1859-1866. Gilmour, R. S. and Paul, J. (1969) J. Mol. Biol. 40, 137-139.

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